Composition containing stilbene glycoside and preparation and uses thereof for treating diabetes

ABSTRACT

Disclosed is a method of processing  Polygonum multiflorum,  the method contains preparing a root extract solution and placing the solution under UV light for 1 to 24 hours. Also disclosed is a composition prepared from the method that can be for use in the prevention, treatment, and management of diabetes in human and animal subjects.

CROSS-REFERENCING

This application claims the benefit of application Ser. No. 14/960,337,filed on Dec. 4, 2015, which is hereby incorporated in reference by itsentireties for all purposes.

FIELD OF THE INVENTION

The invention relates to a composition for use in the prevention,treatment, and management of diabetes in human and animal subjects. Morespecifically, the present invention provides a composition extractedfrom one or more plants of Fallopia genera, and methods of using andpreparing the composition thereof.

BACKGROUND OF THE INVENTION

Diabetes is a one of the leading causes of death in the U.S. and thenumber and percentage of U.S. population with diabetes is rapidlyincreasing. Today, 25.8 million children and adults—8.3% of the U.S.population—have diabetes. The number will reach 366 million by 2030,according to the World Health Organization. the total medical costsannually in the U.S. for diabetes exceeded $100 billion.

There are mainly three types of diabetes: type 1, type 2, andgestational diabetes. Among them, type 2 diabetes, also referred to asnon-insulin dependent diabetes mellitus (NIDDM), represents more than90% of all diabetes patients. Insulinis a hormone that regulates thebody metabolism mainly by promoting the absorption of glucose from theblood to muscles and other tissues. Type 2 diabetes is characterizedwith insulin resistance, which is a diminished ability of insulin toexert its biologic action on regulating glucose. Insulin resistance,related strongly to a sedentary lifestyle, is also associated with avariety of abnormalities including obesity, hypertension,hyperlipidemia, and hyperuricemia. Thus, there is a clear need todevelop diabetes therapies.

However, current therapeutic strategies for diabetes, especially type 2diabetes are limited and major anti-diabetic agents generally sufferfrom inadequate efficacy and high side effects. Side effects fromdiabetes drugs are often severe, which include hypoglycemia, lacticacidosis, idiosyncratic liver cell injury, permanent neurologicaldeficit, digestive discomfort, headache, dizziness, and even death. Forinstance, pioglitazone, rosiglitazone, two widely used diabetes drugs,are found to promote weight gain, a major adverse event associated withthese two drugs' effects on adipose cell differentiation andtriglyceride storage.

There is an urgent and strong need to develop an effective therapy withfew adverse effects for diabetes patients.

is SUMMARY OF THE INVENTION

This invention provides a composition extracted from Fallopia generaplants. As a naturally derived agent, the composition provides a widerrange of utilities for preventing, treating and managing diabetes safelyand effectively.

One aspect of this invention relates to a composition for use in theprevention, treatment, and management of diabetes in human and animalsubjects, comprising 2,3,5,4-tetrahydroxystilbene 2-O-β-glucopyranoside(stilbene glycoside) collected from one or more plants selected from thegroup consisting of Fallopia genera of plants.

The stilbene glycoside can be cis-stilbene glycoside and/ortrans-stilbene glycoside. More specifically, the stilbene glycosidecontains 0-99 wt % cis-stilbene glycoside and 100-1 wt % trans-stilbeneglycoside.

Typically, the cis-stilbene glycoside described above is derived fromthe trans-stilbene glycoside by exposing to UV light.

One example of the Fallopia genera plants is Polygonum multiflorum (PM).

The diabetes treated can be type 2 diabetes.

Another aspect of this invention relates to a method for processing PM.The method is carried out as follows: (i) crushing dried roots of PM topowder; (ii) extracting the PM powder with an ethanol solution under theroom temperature for at least 2 days to obtain an ethanolic extract, theratio of solution to solid being about 1:10 (v/w); (iii) evaporating andconcentrating the ethanolic extract under reduced pressure to obtain adried extract; (iv) subjecting the dried extract to macroporous resinchromatography, followed by eluting the resin with ethanol solutions ofdifferent concentrations to obtain a first eluant; (v) evaporating anddrying the first eluant under reduced pressure to obtain PM extractpowder; (vi) dissolving the obtained PM extract powder in an aqueoussolution to obtain a PM solution; and (vii) placing the PM solutionunder UV light for 1 to 24 hours. An UV-treated PM solution is finallyobtained.

The PM solution discussed above can contain trans-stilbene glycoside ata concentration of 1-50 mg/ml. Preferably, the PM solution containstrans-stilbene glycoside at a concentration of 2-20 mg/ml.

Advantageously, the UV light used has a wavelength of approximately 365nm.

The PM solution can be placed under the UV light for 5-15 hours. Also,it can be placed under the UV light for 5-10 hours.

Further, the UV-treated PM solution contains cis-stilbene glycoside andtrans-stilbene glycoside and the ratio of cis-stilbene glycoside totrans-stilbene glycoside is greater than 1:1. One example of the ratioof cis-stilbene glycoside to trans-stilbene glycoside is between 3:1 and5:1. Another example of the ratio is at or greater than 10:1.

Moreover, the UV-treated PM solution can be treated by HPLC to obtain asecond eluant followed by evaporating and drying under reduced pressureto obtain UV-treated PM extract powder.

The first eluent and the PM extract powder obtained thereafter eachcomprise trans-stilbene glycoside. On the other hand, the second eluantand the UV-treated PM extract powder obtained thereafter each comprisecis-stilbene glycoside.

The HPLC described in step (vii) above is performed under the conditionsthat include a HYPERSIL-C18 column, a mobile phase gradient of 15 minfrom 40% acetonitrile to 65% acetonitrile, and a flow rate of 8 mL/min.

Still another aspect of this invention relates to a composition preparedfrom the above method of processing PM. Consequently, the compositioncontains cis-stilbene glycoside and trans-stilbene glycoside and theratio of cis-stilbene glycoside to trans-stilbene glycoside is greaterthan 1:1. As enumerated above, one example of the ratio of cis-stilbeneglycoside to trans-stilbene glycoside is between 3:1 and 5:1 and anotherexample of the ratio is at or greater than 10:1.

Yet another aspect of this invention relates to a method of extractingcis-stilbene glycoside from one or more plants selected from the groupconsisting of Fallopia genera of plants. The method has the steps of:(i) providing dried roots from one or more plants selected from thegroup consisting of Fallopia genera of plants; (ii) preparing anUV-treated root extract solution using the above method of processingPM; and (iii) purifying cis-stilbene glycoside from the UV-treated rootextract solution, wherein the purification comprises high performancechromatography or recrystallization.

The cis-stilbene glycoside prepared from the just-described method canbe in a composition for prevention, treatment, and management ofdiabetes in human and animal subjects.

Still another aspect of this invention relates to a method for treatmentand management of diabetes. The method includes administering aneffective dose of a composition to a subject in need thereof and thecomposition is prepared from the above method of processing PM.

Examples of the composition are enumerated above, which includecis-stilbene glycoside and/or trans-stilbene glycoside.

Yet another aspect of this invention relates to a method convertingtrans-stilbene glycoside to cis-stilbene glycoside at a conversion rateof at least 50%. The method includes the steps of (i) providing asolution comprising trans-stilbene glycoside at a concentration of 1-50mg/ml; and (ii) placing the solution under UV light having a wavelengthof approximately 365 nm for 1 to 24 hours.

The details of the invention are set forth in the drawing anddescription below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

These drawings illustrate certain aspects of some of the embodiments ofthe present invention, and should not be used to limit or define theinvention.

FIG. 1 is a diagram illustrating a HPLC chromatogram for the PM extract.

FIG. 2 is a bar diagram that shows effect of the PM extract on levels ofIL-6, IL-1b, and TNF-a in liver samples.

FIG. 3 is a diagram showing effect of the PM extract on selected markersfrom insulin signaling pathway.

FIG. 4 is a diagram showing effect of the PM extract on serum insulinlevels of KK CgAy/J mice.

FIG. 5 is a diagram showing approximated insulin resistance (IR) and βcell function (β) from a HOMA model.

FIG. 6 is a diagram that shows effect of stilbene glycoside on serumglucose and insulin levels.

FIG. 7 is a diagram showing effect of stilbene glycoside on (A) glucosetolerance and (B) insulin tolerance.

FIG. 8 illustrates chemical structures of (A) trans-stilbene glycosideand (B) cis-stilbene glycoside.

FIG. 9 shows NMR and LC-MS spectrums of trans-stilbene glycoside andcis-stilbene glycoside.

FIG. 10 includes HPLC chromatograms for the three solutions used in theanimal study to investigate possible anti-diabetic effect ofcis-stilbene glycoside.

FIG. 11 is a bar diagram showing anti-diabetic effects of trans-stilbeneglycoside and cis-stilbene glycoside in different proportions.

FIG. 12 is a diagram showing Anti-diabetic effects of puretrans-stilbene glycoside and cis-stilbene glycoside in high fat dietinduced male CF-1 mice.

FIG. 13 is a diagram showing PEPCK mRNA expression normalized toβ-actin.

DETAILED DESCRIPTION

Polygonum multiflorum (PM), other names He Shou Wu or Fo-ti, is one ofthe Fallopia genera plants. It is used in China as a longevity tonic forgraying hair, premature aging, weakness and other dysfunctions. The rootof PM is used as a tonic and an anti-aging agent in many remedies intraditional Chinese medicine.

The medicinal effects of PM in the treatment of these age-relateddiseases are possibly mediated by the antioxidant capacity of thisplant. In 2005 the extracts of 30 Chinese medicinal plants were studiedsystematically for their antioxidant activities and PM root was found tobe among the highest for both aqueous and methanol extracts. PM extracthas been found both in vitro and in vivo to possess antioxidantactivity. Research indicates that PM enhances the cellular antioxidantactivity, increases the function of superoxide dismutase (SOD),significantly inhibits the formation of oxidized lipids, represses lipidperoxidation in rat heart mitochondria and enhances antioxidant enzymesin the liver.

By analysis technologies such as TLC, HPLC, and LC-MS, many componentsin PM extracts have been identified, which include2,3,5,4-tetrahydroxystilbene-2-O-β-D-glucoside,emodin-8-O-β-D-glucoside, physcion-8-O-β-D-glucoside, emodin,chrysophanol, rhaponticoside, torachrysone-8-O-β-D-glucoside,chrysophanol-8-O-β-D-glucoside, physcion and so on.

2,3,5,4-tetrahydroxystilbene 2-O-β-glucopyranoside (stilbene glycoside)with the chemical structure C₂₀H₂₂O₉ and molecular weight 406.39, is awhite amorphous powder, soluble in water, methanol and ethanol. It isstable in water solutions, but high temperature (>80° C.) might affectits stability; it's very unstable in acid. stilbene glycoside is themajor active compound in PM, and the concentration in the roots of PMcan reach 3%-6%. According to Chinese Food and Drug Administration,stilbene glycoside is used as an index of quality control for PMproducts and the concentration of stilbene glycoside in commercial PMproducts has to be higher than 1%.

stilbene glycoside is a type of stilbene derivative, and otherwell-known stilbene compounds in stilbene family include resveratrol(FIG. 2.1 B) and pterostilbene. The only difference between structuresof stilbene glycoside and resveratrol is the glycoside portion. Becauseof the additional hydroxyl group at the iso-position, stilbene glycosidehas higher antioxidant activity than resveratrol.

Using Ultra-performance liquid chromatography—time-of-flight massspectrometry (UPLC-Q-TOF/MS) and HPLC-UV, the pharmacokinetics,bioavailability, absorption, and metabolism of stilbene glycoside werestudied in rats following a single intravenous or oral administration.It was found that stilbene glycoside was rapidly distributed (within 30min) and then eliminated from rat plasma. Absolute bioavailability ofstilbene glycoside was 40%. Total recovery of unchanged stilbeneglycoside within 24 hr were low (0.041% in bile, 0.06% in feces),whereas the amount of unchanged stilbene glycoside excreted in the urinewithin 24 hr was lower than Lower Limit of Quantification (LLOQ).stilbene glycoside was excreted mainly in the forms of metabolites,including monoglucuronide and the deglycosidated form which is morestable.

Cis-stilbene polyphenols are found to always have higher activities thantheir trans-isomers. Cis-stilbene glycoside was first discovered in PMroots in 2002 and the structure was identified with NMR. The level ofcis-stilbene glycoside was found to be very low in PM, making it veryhard to isolate and enrich. To this date there is rarely any endeavorwhich enriches the level of cis-stilbene glycoside in PM and studies itsactivity. Yet, cis-stilbene polyphenols could be induced withisomerization from trans-stilbenes with UV-light. Cis-stilbene glycosideis an efficacious compound in PM responsible for its anti-diabeticactivities.

EXAMPLES Example 1: Anti-Diabetic Effect of a PM Extract Preparation ofExtract from PM Roots

Dried root powder of PM was purchased from Anguo Mayway Herb CompanyLtd., An Guo, Heibei Province, China, and followed extraction procedureof Lishuang Lv with slight modification. Briefly, the dried roots of PMwere crushed and extracted with 60% ethanol, at a ratio of solution tosolid of 1:10 (v/w), at room temperature for 2 days. The plant materialwas filtered off, and the ethanolic extracts were combined andconcentrated under reduced pressure using a rotary evaporator. The dryextract obtained was then subjected to open column chromatography (CC)packed with macroporous resin. The column was eluted stepwise with eachof 9 different concentrations of ethanol (10%, 20%, 30%, 40%, 50%, 60%,70%, 80% and 90%). The 40% aqueous-ethanol fraction was thenconcentrated under reduced pressure using a rotary evaporator. Thepowder obtained was subjected to HPLC for analysis, and used for animalstudy later.

HPLC Analysis of a PM Extract

PM extract was analyzed by a Waters Acquity HPLC system coupled with aUV detector (Waters, Milford, Mass.). A 250 mm×4.6 mm inner diameter, 5μm, HYPERSIL-C18 column was used. For binary gradient elution, mobilephases A (100% water with 0.2% formic acid) and B (acetonitrile) wereused. The flow rate was maintained at 1 mL/min, and the mobile phasebegan with 8% B. It was followed by progressive linear increases in B to35% at 17 min, to 90% at 18 min, and maintained at 90% until 21 min. Theinjection volume was 10 μL for each sample.

KK CgAy/J Yype 2 Diabetic Mouse Model

Transgenic female KK CgAy/J diabetic mice were purchased from Jacksonlabs (Barr Harbor, Me.) and were housed in stainless steel wire-bottomedcages and acclimatized under laboratory conditions (19-23° C., humidity60%, 12 h light/dark cycle). The mice were divided into two groups with10 each, and fed with Western HFD (Research Diets, New Brunswick, N.J.)composed of 20 kcal % protein, 20 kcal % carbohydrate, and 60 kcal % fat(from butter). Group I: Diabetic control which had free access todrinking water; Group II: Treatment group which had free access todrinking water with 0.075% of PM extract. Body weight, food and wateruptake of the mice were taken on a regular basis. After 7 weeks, all theexperimental mice were sacrificed. Body weight, blood glucose level andlipid profile were recorded. Blood glucose was measured with bloodglucose test strips from Contour, and lipid profile was measured withPTS Panels test strips. The weight of parametrial fat, retro-peritonealfat and brown fat was recorded. Liver, spleen and kidney were removedand weighed as well.

Biochemical Assays

Blood sample was collected and centrifuged at 12,500 rpm for an hour.Serum insulin level was measured with a commercial kit (Cayman Chemical,Detroit, Mich.), and performed according to the protocol. Liver and fattissues were homogenized, lysed with lysis buffer [(0.5% (w/v) sodiumlauryl sarkosinate+10 mM EDTA+0.5 mg/ml proteinase K+0.1 mg/ml RNase Ain 50 mM Tris-Base, pH 8.0)], centrifuged and protein of homogenatesquantified using a BCA protein assay kit (Pierce Chemical, Rockford,Ill.).

ELISA Assay

The levels of Pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in theliver homogenates of control and experimental groups of KK CgAy/J micewere determined by specific ELISA kits according to the manufacturer' sinstructions (Camarillo, Calif.). The capture antibody, diluted withPBS, was used to coat a 96-well plate overnight at room temperature. Theplate was then washed, blocked (1% BSA, 5% sucrose in PBS with 0.05%NaN₃), and washed again. The standards were added to the plate leavingat least one zero concentration well and one blank well. The dilutedsamples (1:5-1:20) were then added to the plate. After incubating for 2h, the plates were washed and the detection antibody was added. Afterincubating for another 2 h the plates were washed and Streptavidin-HRPwas added. After 20 min incubation, the plates were washed, andsubstrate (H₂O₂) and tetramethylbenzidine were added. After another 20min incubation, the stop solution (2 N of H₂SO₄) was added and then,plates were read with a microplate reader at a wavelength of 450 nm.Standard plots were constructed by using standard cytokines and theconcentrations for unknown samples were calculated from the standardplot.

Western Blotting

The sample of liver or fat homogenates (60 μg of protein) in 4× loadingbuffer was denatured at 95° C. for 5 min, and subjected toSDS-polyacrylamide gel (4-10%) electrophoresis. The gel then wastransferred onto a polyvinylidene difluoride membrane (Bio Rad,Hercules, Calif.), and the membrane was blocked with TBS-T (20 mMTris-HCl, 150 mM NaCl, and 0.1% Tween 20, pH 7.4), containing 5-7%nonfat dried milk. The blocked membrane was incubated at 4° C. overnightwith 1:500 dilution of monoclonal antibody for IR-α, IRS-1 (liver) andGlut4 (fat) (Santa Cruz Biotechnology, Santa Cruz, Calif.). Theimmunoblotted membrane was incubated at room temperature for 2 hr withsecondary anti-rabbit or anti-mouse IgG antibodies conjugated withhorseradish peroxidase and then exposed on X-ray film with ECL detectionreagent (Amersham Pharmacia Biotech, Piscataway, N.J.). Bands werequantified using the Adobe Photoshop program with scanning process.

All experiments and analyses were performed at least in triplicate.Results are expressed as means±SE. Statistical analyses were performedusing the Student's T-test. * denotes the difference was statisticallysignificant (p<0.05).

Results (1) Hypoglycemic Effect of PM in KK CgAy/J Diabetic Mice

Results are shown in Table 1 below. After 7 weeks of PM extractadministration in drinking, body weight of transgenic KK Cg/Ay mice wascomparable between control group and treatment group after 7 weeks testperiod. Triglyceride level was significantly higher in the treatmentgroup, but the other lipid parameters, such as HDL, LDL and totalcholesterol almost stayed the same in the two groups. Weight of liverand kidney also showed no difference in the two groups, but spleenshowed a significant weight loss. At the same time, the hyperglycemia oftransgenic diabetic KK Cg/Ay mice was almost completely reverted tonormal by the PM extract treatment, since the glucose level in thecontrol group and treatment group is 233.6 mg/dl and 121.6 mg/dl,respectively. That indicates that the PM extract had a stronghypoglycemic effect.

TABLE 1 Anti-diabetic effects of feeding PM extract in the drinkingwater to female KK CgAy/J mice List of Assays Group 1 Group 2 Bodyweight (g) 40.46 ± 1.91  41.06 ± 0.75  Glucose (mg/dl) 233.56 ± 33.18  121.60 ± 13.07** Triglyceride (mg/dl) 238.44 ± 13.80  271.70 ± 34.59 Total cholesterol (mg/dl) 173.33 ± 3.38  179.30 ± 4.53  HDL (mg/dl)90.00 ± 2.38  86.00 ± 3.07  LDL (mg/dl) 35.62 ± 5.77  39.00 ± 5.96 Parametrial fat (g) 4.73 ± 0.34 4.96 ± 0.31 Retroperitoneal fat (g) 0.58± 0.06  0.75 ± 0.04* Brown fat (g) 0.80 ± 0.07 0.78 ± 0.06 Liver (g) 2.1 ± 0.17 2.09 ± 0.11 Spleen (g) 0.15 ± 0.01  0.12 ± 0.01* Kidney (g)0.34 ± 0.02 0.37 ± 0.01 **P < 0.003, *P < 0.05 as determined by theStudent' T-test. Group 1 (drinking water); Group 2 (0.075% PM extract indrinking water) for 7 weeks.

(2) Pro-Inflammatory Cytokine Levels in the Liver

Pro-inflammatory cytokine (IL-6, IL-1β, and TNF-α) levels in the liverwere measured with commercial Elisa kits after seven weeks of PMtreatment. From FIG. 2, levels of IL-6 and IL-1b didn't differsignificantly between diabetic control group and PM group. However, forTNF-α the level slightly increased in PM treated group (p<0.05). Thelevel of IL-6 was the highest among all three cytokines, at around 200pg/mg.

(3) Markers for Insulin Signaling Pathway

The levels of selected marker from insulin signaling pathway includingIR-α and IRS-1 from the liver and Glut4 from the fat, did not show anysignificant difference from diabetic control and PM group (see FIG. 3),which is consistent with our previous findings. Taken together, theseresults suggest that the hypoglycemic effect of PM extract was notmediated through insulin resistance.

(4) Serum Insulin Levels

Serum insulin level significantly increased from 1 μU/ml in diabeticgroup to around 7 μU/ml in PM extract (FIG. 4), indicating that PMextract had a potent effect in stimulating insulin secretion.

(5) HOMA-β and HOMA-IR

Based on the calculation from Homeostatic Model Assessment (HOMA) model,HOMA-IR=Glucose*Insulin/405 and HOMA-β=(360*Insulin)/(Glucose-63); withfasting blood glucose and fasting blood insulin levels, insulinresistance increased slightly in PM group, but what's more significantis β cell function, which improved by nearly 20 fold from 2 to 45 afterPM treatment.

Example 2: Evaluation of Stilbene Glycoside for its Anti-Diabetic EffectPurification of Stilbene Glycoside

PM roots were extracted in the same manner as described in Example 1,and macroporous resin column was also used for separation. The columnwas eluted with 2 of 40% ethanol to get rid of impurities, and then with1 of 50% ethanol. Fractions collected were monitored with HPLC analysisand the ones with pure stilbene glycoside were piled and concentratedunder reduced pressure using a rotary evaporator. stilbene glycoside wasanalyzed with the same HPLC program as in 1.2.2.

Anti-Diabetic Effect of Stilbene Glycoside in Animal Study

Female KK CgAy/J mice were also utilized in this experiment and handledwith the same protocol as in 1.2.3. The mice were divided into fourgroups with 10 each, and fed with Western HFD. Group I: Normal KKcontrol mice which had free access to drinking water and normal chow;Group II: Diabetic control which had free access to drinking water andHFD; Group III: Treatment group which had free access to drinking waterwith 0.075% of PM-stilbene glycoside and HFD. Group IV: Positive controlgroup which had free access to metformin at 300 mg/Kg and HFD. Bodyweight, food and water uptake of the mice were taken on a regular basis.After 12 weeks, all the experimental mice were sacrificed. Similarmeasurements were taken to the previous experiment with PM extract.

Before sacrifice, all the experimental mice were subjected to glucoseand insulin tolerance tests. For glucose tolerance test, mice werefasted overnight and injected with glucose solution at 1 g/10 ml and 10ml/kg body weight. The change in blood glucose level was monitoredduring the following two hours. For insulin tolerance test, mice werefasted for 5 hours and injected with insulin solution at 1 u/ml and 10ml/kg body weight. The change in blood glucose level was monitoredduring the following two hours.

Results

At the end of the feeding experiment, mice were sacrificed and measuredfor blood glucose and blood insulin levels. Diabetic control KK CgAy/jmice had significantly higher level of blood glucose compared to normalKK mice, and it was effectively brought down by anti-diabetic drugmetformin (300 mg/kg); however, diabetic mice treated with 0.075%stilbene glycoside in drinking water had elevated levels of serumglucose as well as serum insulin compared to diabetic control (see FIG.6), suggesting it did not exert any hypoglycemic effect.

To confirm the absence of hypoglycemic effect of stilbene glycoside,glucose tolerance test and insulin tolerance test were also carried outright before sacrifice. From the result of glucose tolerance test (FIG.7A), 30 minutes after glucose injection (1 g/kg), the blood glucoselevels in all experimental groups increased drastically, with stilbeneglycoside group being the most elevated, and the blood glucose instilbene glycoside group remained much higher than normal control andmetformin groups at the end of the test, suggesting the diabetic mice instilbene glycoside group were glucose intolerant. On the other hand, theinsulin tolerance test displayed that both stilbene glycoside andmetformin groups had higher level of glucose 30 minutes after insulininjection, however, two hours after injection, the blood glucose inmetformin group dropped to a lower level than that of stilbene glycosidegroup (FIG. 7B). The positive control metformin had glucose toleranceand insulin tolerance similar to normal control, while the diabeticcontrol had a high level of intolerance to both glucose and insulin,validating both models.

Example 3: Cis-Stilbene Glycoside as Possible Anti-Diabetic Agent fromPM

It is imperative to develop a model which is convenient, stable, fastand inexpensive, and resembles human type 2 diabetes. The closer it cameto sharing the metabolic characteristics of patients with type 2diabetes, the more relevant and useful it would be in screeningpotential anti-diabetic agents. In the literature C57BL/6 (C57) mice arewidely used in a diet induced model for type 2 diabetes. This strainbecomes obese, hyperglycemic and insulin resistant when fed a HFD and itwas concluded that the high-fat diet-fed C57 mouse model is a robustmodel for impaired glucose tolerance (IGT) and early type 2 diabetes,which may be used for studies on pathophysiology and development of newtreatment. On the other hand, CF-1 mouse is an excellent model forobesity and it has been traditionally employed by our lab to study theanti-obesity effect of a variety of nutraceuticals. However, thesuitability of HFD-induced CF-1 mouse as a model for type 2 diabetes wasnever explored in literature. Once the most appropriate model isidentified, it would be used to test the anti-diabetic effects of puretrans- and cis-stilbene glycoside, and to confirm if the hypoglycemiceffect of PM extract stems from cis-stilbene glycoside.

Purification of Cis-Stilbene Glycoside with Isomerization fromTrans-Stilbene Glycoside

Trans-stilbene glycoside was generated as described in preparation of aPM extract of Example 1. Pure trans-stilbene glycoside was dissolved inlarge volume of water and placed under UV-light over night. The reactionwas monitored with HPLC analysis and the solution after UV exposure wasconcentrated, filtered, and subjected to preparative HPLC. AHYPERSIL-C18 column was used and mobile phase gradient of 40% to 65% B(acetonitrile) over 15 min was chosen. The flow rate was maintained at 8mL/min. Pure cis-stilbene glycoside generated was piled, concentratedwith a rotary evaporator and dried with a freeze drier.

Identification with Nuclear Magnetic Resonance Spectroscopy (NMR) andLiquid Chromatography-Mass Spectroscopy (LC-MS)

¹H NMR and ¹³C NMR spectra were acquired for both trans-stilbeneglycoside and cis-stilbene glycoside on an AMX-500 spectrometer (Bruker,Rheinstetten, Germany) at Department of Chemistry, Rutgers University.For NMR analysis, all compounds were dissolved in CD₃OD.

LC-MS was performed for both isomers with a TIC detector. Prior to LC-MSanalysis, all samples were filtered through a 0.45 μm PTFE syringefilter. ChemStation software (Version 3.01) was used for dataacquisition and analysis. Ionization parameters included: capillaryvoltage, 3.5 kV; nebulizer pressure, 35 PSI; drying gas flow, 10.0mL/min; and drying gas temperature, 350° C. MSD signal parametersincluded: mode, Selected Ion Monitoring (SIM); fragmentor voltage, 70V;gain, 1.0; dwell time, 144 msec; and % relative dwell time, 25.

Animal Study with Trans- and Cis-Stilbene Glycoside Extracts

Female KK mice were purchased from Jackson labs (Barr Harbor, Me.) andwere housed in stainless steel wire-bottomed cages and acclimatizedunder laboratory conditions (19-23° C., humidity 60%, 12 h light/darkcycle). Normal control mice (n=10) were fed with control diet which wasnormal Chow 5001 from LabDiet (St. Louis, Mo.) and was composed of 28.5kcal % protein, 58 kcal % carbohydrate and 13.5 kcal % fat. Diabeticcontrol mice (n=10) were induced with HFD (Research Diets, NewBrunswick, N.J.) composed of 20 kcal % protein, 20 kcal % carbohydrate,and 60 kcal % fat (from butter) for 18 weeks. Three solutions wereadministered to mice (n=10 each) on HFD ad libitum and the compositionsare shown below. Solution 1: pure trans-stilbene glycoside (0.05% indrinking water), HPLC chromatogram in FIG. 3.2 (A); Solution 2: 60%ethanol extract of PM root powders, obtained according to procedure inSection 1.2.1 (proportion of cis-stilbene glycoside to trans-stilbeneglycoside is approximately 1:20, 0.075% in drinking water), HPLCchromatogram in FIG. 3.2 (B); Solutions 3: obtained from exposingsolution 2 under UV-light overnight (proportion of cis-stilbeneglycoside to trans-stilbene glycoside is approximately 2:3, 0.075% indrinking water), HPLC chromatogram in FIG. 3.2 (C). After 18 weeks, bodyweight and blood glucose of all mice were measured.

Screening of Mice Model for Type 2 Diabetes

CF-1 mice and C57BL/6 (C57) mice (male and female) were purchased fromCharles River laboratories (Horsham, Pa.) and were housed in stainlesssteel wire-bottomed cages and acclimatized under laboratory conditions(19-23° C., humidity 60%, 12 h light/dark cycle). Each strain wasdivided into four groups with ten mice each: M-control, male mice onnormal diet; M-HF, male mice on Western HFD; F-control, female mice onnormal diet; F-HF, female mice on Western HFD. All the mice had accessto drinking water ad libitum. Body weight and nonfasting glucose levelswere taken at week 5, 8 and 12. At the end of week 12, all theexperimental mice were sacrificed. Lipid profile was measured with PTSPanels test strips; parametrial fat, retro-peritoneal fat and brown fattissues were harvested and weighed; liver, spleen and kidney wereharvested and weighed as well. Before sacrifice, glucose tolerance testwas performed on all mice as described before.

Animal Study with Pure Trans-Stilbene Glycoside and Pure Cis-StilbeneGlycoside with Male CF-1 Mice

Male CF-1 mice were selected as the model for type 2 diabetes. The micewere kept on Western HFD for 12 weeks and were divided into five groupswith 10 each. Group I: Normal control which had free access to drinkingwater and normal Chow; Group II: Diabetic control which had free accessto drinking water and Western HFD; Group III: Treatment group which hadfree access to drinking water with 0.01% of pure trans-stilbeneglycoside and HFD; Group IV: Treatment group which had free access todrinking water with 0.01% of pure cis-stilbene glycoside and WesternHFD. Group V: Positive control group which had free access to drinkingwater with 0.01% of caffeine and Western HFD. Body weight, food andwater uptake of the mice were taken on a regular basis. After 12 weeks,all the experimental mice were sacrificed. Body weight, blood glucoseand blood insulin levels were evaluated as described before. Glucosetolerance test was also carried out at the end of the study.

PEPCK Assay with Pure Trans-Stilbene Glycoside and Pure Cis-StilbeneGlycoside

Aside from animal study, both trans- and cis-stilbene glycoside will beevaluated for their effects on phosphoenolpyruvate carboxykinase(PEPCK), which is the key enzyme catalyzing the first step in hepaticgluconeogenesis. Glucagon and stress hormones, such as glucocorticoids,upregulate PEPCK gene expression in hepatocytes via a cyclic AMP(cAMP)-dependent pathway. Alternatively, insulin strongly repressesPEPCK transcription through the activation of the phosphoinositide-3kinase (PI3K) pathway 170. Normally, the increase in blood glucoselevels after food intake stimulates the secretion of insulin from thepancreas. This increase in blood insulin concentration then leads to thedown-regulation of PEPCK gene expression and, subsequently, thecessation of gluconeogenesis by the liver. Insulin resistanthepatocytes, however, are unable to effectively convey the insulinsignal, leading to an increase in PEPCK mRNA transcription 171. Thus,the glucose synthesis persists despite a high blood glucoseconcentration. The compounds that are able to repress PEPCK expressionand overcome insulin resistance could constitute a new class of glucoselowering agents 172.

The HepG2 cells were plated in 24-well tissue culture plates and weregrown to near confluence in Dulbecco's modified Eagle's mediumcontaining 2.5% (vol/vol) newborn calf serum and 2.5% (vol/vol) fetalcalf serum. Cells were treated for 8 h with 500 nM dexamethasone and 0.1mM 8-CTP-cAMP (Dex-cAMP) to induce PEPCK gene expression together withtest compounds (5 μM trans-stilbene glycoside and 5 μM cis-stilbeneglycoside).

Total RNA was extracted from HepG2 cells using Trizol reagent, followingthe manufacturer's instructions. RNA was quantifiedspectrophotometrically by absorbance measurements at 260 and 280 nm.Quality of RNA was assessed by separation in gel electrophoresis. RNAwas then treated with Dnasel (Invitrogen), following the manufacturer'sguidelines, to remove any traces of DNA contamination. The cDNAs weresynthesized with 2.5 g of RNA for each sample, using Stratascriptreverse transcriptase (Stratagene, La Jolla, Calif.), following themanufacturer's protocol. The synthesized cDNAs were diluted fourfold.Five microliters of each of these diluted samples was used for PCRreactions of 25 ηL final volume. The other components of the PCRreactions were 0.5 ηL of 6 ηM gene-specific primers and 12.5 ηL ofBrilliant SYBR Green PCR master mix (containing green jump-start Taqready mix). ROX (Stratagene, La Jolla, Calif.) was used as a referencedye. The primers were selected using the Primer Express version 2.0software (Applied Biosystems, Foster City, Calif.). These primersgenerated a 76-bp product from β-actin mRNA. The intron-spanning forwardprimer was selected to cover the exon 9-10 boundary. The reverse primerwas selected from exon 10. The oligos were synthesized by IDT. Theseprimers generated a 74-bp product from PEPCK mRNA and a 207-bp productfrom genomic DNA.

Quantitative PCR (qPCR) amplifications were performed on an MX3000psystem (Stratagene, La Jolla, Calif.) using one cycle at 50° C. for 2min and one cycle of 95° C. for 10 min, followed by 40 cycles of 15 s at95° C. and 1 min at 60° C. The dissociation curve was completed with onecycle of 1 min at 95° C., 30 s at 55° C., and 30 s at 95° C. Non-RTcontrol and no-template control were included in each experiment asquality control steps.

PEPCK mRNA expressions were analyzed using the ΔΔCT method andnormalized with respect to the expression of the β-actin house keepinggene. The ΔΔCT Values obtained from these methods reflect the relativemRNA quantities for a specific gene in response to a treatment asrelative to a calibrator. The Dex-cAMP treatment (positive control)served as the calibrator sample in this study. The PECPK gene expressionof the calibrator sample was assigned to a value of 1.0. A value of <1.0indicates transcriptional down-regulation (inhibition of geneexpression) relative to the calibrator. Amplification of specifictranscripts was further confirmed by obtaining melting curve profiles.All samples were run in duplicate.

Results (1) NMR and MS for Trans- and Cis-Stilbene Glycoside

Trans-stilbene glycoside and cis-stilbene glycoside were identified with¹H and ¹³C NMR spectra as well as LC-MC spectrum by comparing toliterature, structures shown in FIG. 8. For trans-stilbene glycoside,¹H-NMR (CD₃OD, 500 MHz) δ: 3.34-3.79 (6H, m, sugar H), 4.50 (1H, d,J=7.8 Hz, H-1″), 6.24 (1H, d, J=2.8 Hz, H-6), 6.61 (1H, d, J=2.8 Hz,H-4), 6.75 (2H, d, J=6.7 Hz, H-3′,5′), 6.91 (1H, d, J=16.4 Hz, H-b),7.44 (2H, dd, J=1.9, 6.7 Hz, H-2′,6′), 7.70 (1H, d, J=16.4 Hz, H-a);¹³C-NMR (CD₃OD, 500 MHz).

For cis-stilbene glycoside, ¹H-NMR (CD₃OD, 500 MHz) δ: 3.39-3.82 (6H, m,sugar H), 4.58 (1H, d, J=7.6 Hz, H-1″), 6.15 (1H, d, J=2.8 Hz, H-6),6.24 (1H, d, J=2.8 Hz, H-4), 6.50 (1H, d, J=12.2 Hz, H-a), 6.62 (2H, d,J=8.5 Hz, H-3′,5′), 6.73 (1H, d, J=12.2 Hz, H-b), 7.08 (2H, d, J=8.5 Hz,H-2′,6′); ¹³C-NMR (CD₃OD, 500 MHz).

The ¹H-NMR spectrum of cis-stilbene glycoside was very similar to thatof trans-stilbene glycoside, with the exception of the couplingconstants of the vinylic protons signals (H-a and H-b), indicating thepresence of two cis-coupled vinylic protons at δ6.71 (H-b) and 6.47(H-a). The ¹³C-NMR spectrum of cis-stilbene glycoside exhibited twochemically equivalent aromatic carbons at δ_(c) 131.4 (C-2′/C-6′) andδ_(c) 115.9 (C-3′/C-5′).

The negative mass spectrum of trans-stilbene glycoside and cis-stilbeneglycoside showed a [M-1]⁻ion at m/z 405. The fragment ion peak m/z 405generated a main fragment ion [M-glc-1]⁻at m/z 243, which can beconsidered characteristic of the presence of an aglycone moiety.

Anti-Diabetic Effect of Extracts of Trans- and Cis-Stilbene Glycoside

Female KK mice were fed with Western HFD for 18 weeks to induce a modelfor type 2 diabetes. Three solutions were employed in the study: puretrans-stilbene glycoside (0.05% in drinking water), PM extract withtrans-stilbene glycoside/cis-stilbene glycoside 1:20 (0.075% in drinkingwater) and PM extract enriched with cis-stilbene glycoside(trans-stilbene glycoside/cis-stilbene glycoside 2:3, 0.075% in drinkingwater), HPLC chromatograms as shown in FIG. 10. Body weight and bloodglucose levels were monitored. From the results, none of the threesolutions significantly decreased high fat induced body weight gaincompared to diabetic control. As for blood glucose, it showed adecreasing trend of blood glucose with increasing level of cis-stilbeneglycoside in the extract. See FIG. 11. And the only solution whichshowed a significant hypoglycemic effect was the cis-stilbene glycosideenriched extract (p<0.05), while the other two did not. Considering thesolution in group 3 was obtained from overnight exposure of solution ingroup 2 under UV-light and the only difference between the two extractsis the level of cis-stilbene glycoside, this data suggested possibleanti-diabetic effect of cis-stilbene glycoside. However this effectneeds to be confirmed by carrying out animal studies with pure trans-and cis-stilbene glycoside. In the present study HFD induced female KKmouse model was utilized to reduce cost of transgenic KK CgAy/j mice,however due to limited supply of KK mice and the length of time it takesfor KK mice to develop obesity and hyperglycemia, it was necessary toexplore other options for a suitable type 2 diabetes animal model whichis economical, reliable and mimics human type 2 diabetes.

(3) Identifying a Model for Type 2 Diabetes

Two strains of mice were identified as possible candidates for dietinduced diabetes model: C57BL/6J (C57) mice and CF-1 mice, and both maleand female mice were evaluated. All mice were fed either a high-fat diet(58% energy by fat) or a normal diet (11% energy by fat), and bodyweight and blood glucose levels were taken at weeks 5, 8 and 12; glucosetolerance test also evaluated at the end of the study.

For C57 strain, both female and male mice had higher body weight at week12 when fed a HFD compared to those on normal diet: F-HF group was 40.2%heavier than F-control while M-HF group was 11.8% heavier compared withM-control. Circulating blood glucose displayed a slight increasing trendfrom week 5 to week 8 except for M-HF, which decreased from 172.10 to156.33 mg/dL; not much difference in blood glucose was shown from week 8to week 12 except for M-control. For glucose tolerance test M-controlgroup was missing because male C57 mice on normal diet displayedaggressive behaviors and by the end of study they were either injured ordead. Both male and female mice on HF diet displayed significant glucoseintolerance since 30 minutes after glucose injection blood glucoserapidly rose to 266.78 and 256.50 mg/dL, respectively, nearly doublingthe level before glucose injection. For lipid profile, not muchdifference was shown in all groups for the three parameters:triglycerides, HDL and LDL, except for triglycerides in M-HF group whichwas slightly higher than M-control, 64.67 versus 50 mg/dL. Body fatincluding parametrial fat and retro-peritoneal fat were much heavier inHF groups compared to control groups, and greater fat accumulation wasobserved in female mice. Weight of important organs, including pancreas,liver and kidney, were slightly higher in HF groups.

CF-1 mice became very obese at the end of 12 weeks, with the weight ofM-HF mice being the highest of all, 53.4% heavier than F-control and13.4% heavier than M-control. For nonfasting glucose, M-HF displayed ahigh level as early as week 5 and remained the highest throughout thecourse of 12 weeks, while F-HF had no significant difference in bloodglucose from the control group. It was also M-HF group that displayedthe highest degree of glucose intolerance: 30 minutes after glucoseinjection, blood glucose jumped to 358.2 mg/dL compared to an initialreading of 134.7 mg/dL, significantly higher than other three groups aswell; after 120 minutes, it remained elevated at 192 mg/dL. The trendfor lipid profile, fat mass and organ mass in CF-1 mice was similar tothat of C57 mice. All information taken together, male CF-1 mice on HFdiet was selected as the model for type 2 diabetes, as it ischaracterized with obesity, elevated blood sugar and a high level ofglucose intolerance, which is very similar to human type 2 diabetes, andis absent for any aggressive behaviors.

(4) Anti-Diabetic Effect of Pure Trans- and Cis-Stilbene Glycoside

After identifying diet-induced male CF-1 mice as the model animal, andobtaining cis-stilbene glycoside from isomerization of trans-stilbeneglycoside and purification with Prep-HPLC, the anti-diabetic effects ofpure cis- and trans-stilbene glycoside was tested in the new model, andcaffeine was used as a positive control. As evident in FIG. 12, bothisomers of stilbene glycoside (0.01% in drinking water) couldsignificantly decrease the serum glucose level in the male CF-1 miceafter HFD treatment for 12 weeks, similar to that of caffeine. In GTT,HF group showed glucose intolerance test since blood glucose increasedsharply 30 min after the intraperitoneal injection of glucose solutionand remained at a very high level after 120 min. In contrast, the risein blood glucose level was greatly suppressed by cis-stilbene glycoside;however, the effect on glucose intolerance was absent in trans-stilbeneglycoside treated group. HF mice had a much higher level of seruminsulin than normal control at the end of the study and in both stilbeneglycoside treatment groups insulin levels were significantly lowered.After calculating the HOMA-IR with the equationHOMA-IR=Glucose*Insulin/405 ¹⁵⁹, insulin resistance was found to begreatly elevated in HF group but reduced in all experimental groups.Calculated HOMA-IR in cis-stilbene glycoside group was 102.7% lower thanthat in trans-stilbene glycoside group, indicating cis-stilbeneglycoside had a much stronger effect in alleviating insulin resistancethan trans-stilbene glycoside.

(5) PEPCK Assay

Real-time PCR, also called quantitative PCR or qPCR, can provide asimple and elegant method for determining the amount of a targetsequence or gene that is present. PEPCK assay was performed with liverHepG2 cells and the level of PEPCK mRNA from real-time PCR is normalizedwith β-actin. Dex/cAMP could induce the transcription of PEPCK gene andas seen in FIG. 13A, both cis-stilbene glycoside and trans-stilbeneglycoside could effectively suppress the up-regulation. However nodose-dependent pattern was observed, as cis-stilbene glycosidesuppressed more efficiently at 30 μM than at 100 μM, while fortrans-stilbene glycoside higher concentration resulted in lower level ofPEPCK transcription. This agrees with the results from the comparativeCt method, where cis-stilbene glycoside (30 μM) had a 61.7% reductioncompared to Dex/cAMP group while trans-stilbene glycoside (100 μM) had53.3% reduction. Therefore cis-stilbene glycoside was found to be moreeffective than trans-stilbene glycoside in ameliorating Dex/cAMP-inducedPEPCK transcription in HepG2 cells.

Example 4. The Extraction of 2,3,5,4′-Tetrahydroxystilbene2-O-β-Glucopyranoside Stilbene Glycoside from Foti (PolygonumMultiflorum or Heshouwu

Extraction: The root of Polygonum multitflorum (PM) was purchased from acommercial medicinal store located in Huanggang city, Hubei province ofChina. There are two types of root available: raw foti root andprocessed foti root. Raw root has a pale white color and is directlyfrom the sundry after washing and cleaning. The processed foti isprocessed from the raw root: i.e. the raw root was washed and cleanedfirst then repeated steam and sundry (at least 4 steam/sundry cycles).Both types of foti are commercially available. The purchased foti rootsamples were dried in an oven at 50° C. for 12 hour, then ground to finepowder. The resulted powder was stored in a dry and dark container. 10 gof foti root powder was weighed and placed in a brown round bottomflask. To the flask, 100 ml of 60% aqueous ethanol was added for theextraction (solid:liquid ratio=1:10). The extraction was conducted atambient temperature for specified time on Table 2 (1-5 days). Upon thefinish of the extraction, the mixture was filtered in vacuo and thesolid waste was washed with 40 ml of 60% aqueous ethanol. The filtratewas combined and concentrated in vacuo to yield a solid sample.

TABLE 2 The influence of different varieties and different soaking timeon the extraction yield Area on Concentration Weight of residueExtraction rate Types and time stilbene glycoside HPLC Total area(mg/mL) (g) (%) Raw foti cis-stilbene 11072 194994 0.0099 2.63 2.60 (1day) glycoside trans-stilbene 183922 glycoside cis-stilbene 12250glycoside Raw foti trans-stilbene 181072 193322 0.0098 3.08 3.03 (2 day)glycoside cis-stilbene 13367 glycoside Raw foti trans-stilbene 240134253501 0.0117 2.72 3.17 (3 day) glycoside Raw foti cis-stilbene 15695358117 0.0148 3.06 4.54 (4 day) glycoside trans-stilbene 342422glycoside cis-stilbene 3171 glycoside Raw foti trans-stilbene 272422275593 0.0123 2.95 3.63 (5 day) glycoside cis-stilbene 14878 glycosideProcessed trans-stilbene 204764 219642 0.0106 2.37 2.52 foti (1 day)glycoside cis-stilbene 16516 glycoside Processed trans-stilbene 475437491953 0.0189 2.8 5.29 foti (2 day) glycoside cis-stilbene 38358glycoside Processed trans-stilbene 109215 147573 0.0084 2.76 2.33 foti(3 day) glycoside cis-stilbene 13015 glycoside Processed trans-stilbene173662 186877 0.0096 2.52 2.43 foti (4 day) glycoside cis-stilbene 16520glycoside Processed trans-stilbene 209463 225983 0.0108 2.78 3.01 foti(5 day) glycoside

Example 5. The Extraction Rate at Different Time for Raw Foti

As illustrated in Table 2, raw foti was immersed in water. The ratio ofsolid to solvent (water) was 1:10. Samples were collected at differentdays, at day 1, day 2, days 3, day 4 and day 5. When the extractionprocess was stopped, the mixture was filtered and residue was washed.The filtrate was combined and concentrated, dried and weight. Analyticalsamples were collected for each extraction sample. The results, i.e.content of both trans-stilbene glycoside and cis-stilbene glycoside ineach raw foti sample, conversion rate from trans-stilbene glycoside tocis-stilbene glycoside, were summarized in Table 2.

Conclusion: for raw foti, the optimum immersement time is 4 days, tohave a maximum extraction rate of stilbene glycoside, at the highestextraction rate of 4.5% of total stilbene glycoside.

Example 6. The Extraction Rate at Different Time for Processed Foti

As illustrated in Table 2, both processed foti were immersed in water.The ratio of solid to solvent (water) was 1:10. Samples were collectedat different days from day 1, day 2, days 3, day 4 to day 5. When theextraction process was stopped, the mixture was filtered and residue waswashed. The filtrate was combined and concentrated, dried and weight.Analytical samples were collected for each extraction sample. Theresults, i.e. content of both trans-stilbene glycoside and cis-stilbeneglycoside in each processed sample, conversion rate from trans-stilbeneglycoside to cis-stilbene glycoside, were summarized in Table 2.

Conclusion: for processed foti, the optimum immersement time is 2 days,to have a maximum extraction rate of stilbene glycoside, at the highestextraction rate of 5.29% of total stilbene glycoside.

Example 7. Purification and Isolation of Stilbene Glycoside From (theAbove) Extracted Solid Residue

One g of dried solid residue obtained above (Example 4) was suspended in10 ml of water solution. The solution was applied to an HP-20(microporous resin) packed liquid chromatography. The following aredetailed experimental procedure.

Microporous resin HP-20 was activated prior to use according tomanufacture's instruction. The obtained dried extract (solid residuefrom Example 4) was then subjected to column chromatography packed withmacroporous resin (HP-20). The column was eluted with a series of mixedsolvents of ethanl and water. The ethanol content is from 0% to 60% inwater. Therefore, the eluting process was eluted with water, 10%ethanol, 20%, 30%, 40%, and 50% followed by 60% aqueous ethanol.Fractions of eluted solution were collected to different flaskcontainers. Each fraction was analyzed with an HPLC analysis and theones with pure trans-stilbene glycoside were combined and concentratedin vacuo and then lyophilized. The purity of trans-stilbene glycosidethus isolated from the liquid chromatography with macroporous resinstationary phase and aqueous ethanol mobile phase was over 99% asdetermined by HPLC and ¹H NMR.)

Example 8. Effect of Different Concentrations of Ethanol Solution on theElution of Stilbene Glycoside

The collected samples from above (Example 4) were analyzed with an HPLCthat has established standard curves of both trans-stilbene glycosideand cis-stilbene glycoside using pure trans-stilbene glycoside andcis-stilbene glycoside. The analysis results obtained from HPLCmeasurement for different solvents used in eluting trans-stilbeneglycoside showed that only three solvent mixture containedtrans-stilbene glycoside: 30%, 40% and 50%. The fraction from 40%aqueous ethanol has the maximum content of trans-stilbene glycoside witha purity of above 98%.

Conclusion: 40% aqueous ethanol is the optimum solvent to elutetrans-stilbene glycoside from the mixture of extracted solid from thepowder of foti.

Example 9. The Influence of Time on the Transformation fromTrans-Stilbene Glycoside to Cis-Stilbene Glycoside under 365 nm UVWavelength

Forty mg of trans-stilbene glycoside was dissolved in 2 mL of dd water(double distilled water) under UV-light (365 nm). The reaction wasmonitored with HPLC (high performance liquid chromatography) analysis.Analytical samples were collected at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 hand 15 h, respectively. The collected samples were analyzed by HPLC withstandard curves to quantify both trans-stilbene glycoside andcis-stilbene glycoside accurately. The transformation rate fromtrans-stilbene glycoside to cis-stilbene glycoside was calculated basedon the standard curve on the HPLC. The results were shown in Table 3.

Conclusion: Under 365 nm UV irradiation, the maximum conversion rate is95% and the time reached the highest conversion rate is at 7 h. At 5 h,the conversion rate is reached 94%.

TABLE 3 Relationship between conversion and irradiation time under 365nm UV light exposure Ratio of cis- stilbene Time glycoside under Area totrans- 365 nm stilbene from stilbene Total Rate of UV glycoside HPLCglycoside area conversion 0 h cis-stilbene 5288  1:99 528821 0%glycoside trans- 523533 stilbene glycoside 1 h cis-stilbene 464255 88:12 527563 88% glycoside trans- 63308 stilbene glycoside 2 hcis-stilbene 470384  89:11 528521 89% glycoside trans- 58137 stilbeneglycoside 3 h cis-stilbene 474926  90:10 527695 90% glycoside trans-52769 stilbene glycoside 4 h cis-stilbene 477356 91:9 524567 91%glycoside trans- 47211 stilbene glycoside 5 h cis-stilbene 495340 94:6526957 94% glycoside trans- 31617 stilbene glycoside 6 h cis-stilbene495001 94:6 526597 94% glycoside trans- 31596 stilbene glycoside 7 hcis-stilbene 500165 95:5 526489 95% glycoside trans- 26324 stilbeneglycoside 15 h  cis-stilbene 497512 95:5 523697 95% glycoside trans-26185 stilbene glycoside

Example 10. The Influence of Time on the Transformation fromTrans-Stilbene Glycoside to Cis-Stilbene Glycoside Under 254 nm UVWavelength

Forty mg of trans-stilbene glycoside was dissolved in 2 mL of dd water(double distilled water) under UV-light (254 nm). The reaction wasmonitored with HPLC analysis. Analytical samples were collected at 1 h,2 h, 3 h, 4 h, 5 h, 6 h, 7 h and 15 h, respectively. The collectedsamples were analyzed by HPLC with standard curves to quantify bothtrans-stilbene glycoside and cis-stilbene glycoside accurately. Thetransformation rate from trans-stilbene glycoside to cis-stilbeneglycoside was calculated based on the standard curve on the HPLC. Theresults were shown in Table 4.

Conclusion: Under 254 nm UV irradiation, the maximum conversion rate is95% and the time reached the highest conversion rate is at 15 h. At 5 h,the conversion rate is reached 88%.

TABLE 4 Relationship between conversion and irradiation time under 254nm UV light exposure Time Ratio of cis- under Area stilbene glycoside254 nm stilbene from to trans-stilbene Total Rate of UV glycoside HPLCglycoside area conversion 0 h cis-stilbene 5288  1:99 528821 0 glycosidetrans- 523533 stilbene glycoside 1 h cis-stilbene 290654 55:45 52846155% glycoside trans- 237807 stilbene glycoside 2 h cis-stilbene 39348875:25 524651 75% glycoside trans- 131163 stilbene glycoside 3 hcis-stilbene 419650 80:20 524563 80% glycoside trans- 104913 stilbeneglycoside 4 h cis-stilbene 447307 85:15 526243 85% glycoside trans-78936 stilbene glycoside 5 h cis-stilbene 464518 88:12 527861 88%glycoside trans- 63343 stilbene glycoside 6 h cis-stilbene 471187 90:10523541 90% glycoside trans- 52354 stilbene glycoside 7 h cis-stilbene491106 94:6  522453 94% glycoside trans- 31347 stilbene glycoside 15 h cis-stilbene 495200 95:5  521263 95% glycoside trans- 26063 stilbeneglycoside

Example 11. The Influence of UV Wavelength on the Transformation fromTrans-Stilbene Glycoside to Cis-Stilbene Glycoside

By comparing Table 3 and Table 4, the two sets of data, it can beconcluded that the conversion rate of the stilbene glycoside (fromtrans-stilbene glycoside to cis-stilbene glycoside) solution irradiatedwith 365 nm ultraviolet light is always higher than that of the stilbeneglycoside irradiated by ultraviolet light at 254 nm. Moreover, the timeneeded for the cis-trans isomerization to reach a balance is an importedfactor for the conversion. The time is much shorter to reach theisomerization balance at the irradiation of 365 nm UV wavelength,indicating that at 365 nm UV irradiation, the conversion is faster andmore effective. For instance, the standard trans-stilbene glycosidesolution irradiated with 365 nm ultraviolet light takes about 7 hours toachieve the optimal conversion, whereas, the standard solutionirradiated with 254 nm ultraviolet light takes about 15 hours to reachthe optimal conversion.

Conclusion: in terms of conversion rate and effectiveness fromtrans-stilbene glycoside to cis-stilbene glycoside, 365 nm is theoptimal UV wavelength. At 5 hours, it reached the conversion of 94% ofcis-stilbene glycoside from 1% or less of cis-stilbene glycoside at thebeginning, whereas, at 254 nm at 5 hours, the maximum conversion is 88%of cis-stilbene glycoside.

Example 12. Purification of Cis-Stilbene Glycoside with Preparative HighPerformance Chromatography

Pure trans-stilbene glycoside (0.2 g) was dissolved in water (20 ml) andplaced under UV-light (365 nm) for 5 hours. The reaction was monitoredwith HPLC analysis. The solution after UV exposure was concentrated,filtered, and subjected to purification of cis-stilbene glycoside with asemi-preparative HPLC (Gilson Inc., Madison, WI) equipped with aHypersil-C18 column (20×300 mm, 10 μm, Fisher) and mobile phase ofacetonitrile and water. Pure fractions of cis-stilbene glycosidegenerated was piled, concentrated in vacuo and lyophilized to yield 0.12g of. cis-stilbene glycoside as a pale white solid. The puritycis-stilbene glycoside thus prepared was over 99% as determined by HPLCand ¹H NMR.

Example 13. Purification of Cis-Stilbene Glycoside with RecrystalizationMethod

Pure trans-stilbene glycoside (2 g) was dissolved in water (50 ml) andplaced under UV-light (365 nm) for 5 hours. The reaction was monitoredwith HPLC analysis. The solution after UV exposure was concentrated,filtered, and subjected to recrystalization as detailed in thefollowing. The solid residue after concentration was re-dissolved inwater. The resulted solution was heated to close to the boiling point ofwater. Then ethanol was added to the heated solution with much care.When white precipitates were formed and the solution turned to cloudy,small amount of ethanol was added while heating. Addition of ethanol wascontinued till the solution turned to clear. The solution was coolednaturally to ambient temperature and then was placed to a refrigerator.The precipitate of the cis-stilbene glycoside was collected. The aboverecrytalization procedure was repeated. The collected cis-stilbeneglycoside product was dried in a heated oven (40 oC) for 18 hours toyield 1.5 g of. cis-stilbene glycoside as a pale solid. The puritycis-stilbene glycoside thus prepared was over 99% as determined by HPLCand ¹H NMR.

Conclusion: Recrystalization method was used for the first time inpurifying cis-stilbene glycoside. Other recrystalization solvent systemother than ethanol-water (Example 9) can be methanol/water;isopropyl/water; acetonitrile/water etc.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

Further, from the above description, one skilled in the art can easilyascertain the essential characteristics of the present invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A method of processing Polygonum multiflorum(PM), the method comprising: crushing dried roots of PM to powder;extracting the PM powder with an ethanol solution under the roomtemperature for at least 2 days to obtain an ethanolic extract, theratio of solution to solid being about 1:10 (v/w); evaporating andconcentrating the ethanolic extract under reduced pressure to obtain adried extract; and subjecting the dried extract to macroporous resinchromatography, followed by eluting the resin with ethanol solutions ofdifferent concentrations to obtain a first eluant; evaporating anddrying the first eluant under reduced pressure to obtain PM extractpowder; dissolving the obtained PM extract powder in an aqueous solutionto obtain a PM solution; and placing the PM solution under UV light for1 to 24 hours, whereby an UV-treated PM solution is obtained.
 2. Themethod of claim 1, wherein the PM solution comprisestrans-2,3,5,4-tetrahydroxystilbene 2-O-β-glucopyranoside (stilbeneglycoside) at a concentration of 1-50 mg/ml.
 3. The method of claim 1,wherein the PM solution comprises trans-2,3,5,4-tetrahydroxystilbene2-O-β-glucopyranoside (stilbene glycoside) at a concentration of 2-20mg/ml.
 4. The method of claim 2, wherein the UV light has a wavelengthof approximately 365 nm.
 5. The method of claim 4, wherein the PMsolution is placed under the UV light for 5-15 hrs.
 6. The method ofclaim 1, wherein the UV-treated PM solution comprises cis-stilbeneglycoside and trans-stilbene glycoside, the ratio of cis-stilbeneglycoside to trans-stilbene glycoside being greater than 1:1.
 7. Themethod of claim 6, wherein the ratio of cis-stilbene glycoside totrans-stilbene glycoside is between 3:1 and 5:1.
 8. The method of claim6, wherein the ratio of cis-stilbene glycoside to trans-stilbeneglycoside is at or greater than 10:1.
 9. A method of extractingcis-stilbene glycoside from one or more plants selected from the groupconsisting of Fallopia genera of plants, comprising: providing driedroots from one or more plants selected from the group consisting ofFallopia genera of plants; preparing an UV-treated root extract solutionusing the method of claim 1; and purifying cis-stilbene glycoside fromthe UV-treated root extract solution, wherein the purification compriseshigh performance chromatography or recrystallization.
 10. A compositionfor use in the prevention, treatment, and management of diabetes inhuman and animal subjects, wherein the composition comprisescis-stilbene glycoside prepared from the method of claim
 9. 11. Acomposition for use in the prevention, treatment, and management ofdiabetes in human and animal subjects, wherein the composition isprepared from the method of claim
 1. 12. The composition of claim 11,wherein the composition comprises cis-stilbene glycoside andtrans-stilbene glycoside, the ratio of cis-stilbene glycoside totrans-stilbene glycoside being greater than 1:1.
 13. The composition ofclaim 12, wherein the ratio of cis-stilbene glycoside to trans-stilbeneglycoside is between 3:1 and 5:1.
 14. The composition of claim 12,wherein the ratio of cis-stilbene glycoside to trans-stilbene glycosideis at or greater than 10:1.
 15. A method for treatment and management ofdiabetes comprising administering an effective dose of a composition toa subject in need thereof, wherein the composition is prepared from themethod of claim
 1. 16. The method of claim 15, wherein the compositioncomprises cis-stilbene glycoside and trans-stilbene glycoside, the ratioof cis-stilbene glycoside to trans-stilbene glycoside being greater than1:1.
 17. The method of claim 16, wherein the ratio of cis-stilbeneglycoside to trans-stilbene glycoside is between 3:1 and 5:1.
 18. Themethod of claim 16, wherein the ratio of cis-stilbene glycoside totrans-stilbene glycoside is at or greater than 10:1.
 19. A method fortreatment and management of diabetes comprising administering aneffective dose of a composition to a subject in need thereof, whereinthe composition comprises cis-stilbene glycoside prepared from themethod of claim
 9. 20. A method of converting trans-stilbene glycosideto cis-stilbene glycoside at a conversion rate of at least 50%,comprising: providing a solution comprising trans-stilbene glycoside ata concentration of 1-50 mg/ml; and placing the solution under UV lighthaving a wavelength of approximately 365 nm for 1 to 24 hours.